The present invention relates generally to sulfone phosphonate compounds and their use in organic synthesis and, more particularly, to disulfone bis-phosphonates and their preparation and use as reagents in the synthesis of biologically relevant disulfones.
Enzymes are biological catalysts which mediate the great majority of biochemical reactions that occur in living organisms. Enzymatically catalyzed reactions typically result in higher reaction rates, under milder reaction conditions, with much greater reaction specificity than other chemical reactions. The specific geometric configuration and identity of the chemical elements that create the reacting group of a reactant or substrate for a particular enzyme are important factors affecting whether that enzyme can catalyze a given reaction. Typically, the amino acids which constitute the enzyme""s substrate-binding site form a pocket, or cleft, in the surface of the enzyme which is geometrically and electronically complementary to the particular shape and charge distribution of the substrate""s functional groups. Thus, a substrate having the wrong charge distribution, stereochemistry, chirality, et cetera will not fit into the enzymatic binding site.
The highly specific nature of enzyme-substrate binding renders enzymatic reactions particularly susceptible to influence by other substances. Compounds that can combine with an enzyme and/or its substrate may affect either substrate binding or the enzyme""s turnover rate. A compound which reduces enzymatic activity by either of these methods is referred to as an inhibitor. In many cases, enzyme inhibitors structurally resemble an enzyme""s natural substrate in at least some respects, but the reaction catalyzed by the enzyme when it is bound to its natural substrate either cannot achieve its normal product or will do so at a considerably reduced rate. Such inhibitors are frequently referred to as analogs. Inhibitors can act through several mechanisms, two of which are competitive inhibition and noncompetitive inhibition.
A compound can be a competitive inhibitor of an enzyme if that compound competes directly with a natural substrate for an enzyme""s binding site. Structurally, this type of inhibitor usually is sufficiently similar to the normal or natural substrate to enable binding to the enzyme active site, but the inhibitor differs from the natural substrate in that it is comparatively unreactive when bound to the enzyme. Since most competitive inhibitors reversibly bind their target enzyme, such compounds tend to reduce the cellular concentrations of free enzyme available for natural substrate binding, thereby inhibiting productive enzymatic activity and reducing the availability of enzyme reaction products. Inhibitors which irreversibly bind an enzyme active site are referred to as inactivators, or suicide inhibitors, and their effects on free enzyme concentration levels are much longer lived.
A substance may be deemed a noncompetitive inhibitor of an enzyme if the substance can bind the enzyme-substrate complex directly but cannot bind the free enzyme. This inhibitory mechanism likely functions by distorting the structure of the active site and rendering the enzyme incapable of catalyzing the reaction with the substrate. A noncompetitive inhibitor need not resemble the substrate at all, for it has no affect on an enzyme""s ability to bind the natural substrate. As such, the noncompetitive inhibitor acts by interfering with an enzyme""s catalytic function, not its ability to bind its natural substrate. Since substrate binding is relatively unaffected, this type of inhibition is thought to occur more frequently in the case of multisubstrate enzymes, such as transferase enzymes which catalyze reactions that transfer a specific functional group from one substrate to another.
As enzymatic reactions often play an important role in a variety of biochemical pathways that affect biological systems, enzymes frequently are good targets for strategic efforts to affect disease processes, such as cancer, or viral invasions of a host system, such as by the human immunodeficiency virus (HIV). For example, small cell lung cancer (SCLC), a highly malignant carcinoma that is prevalent in cigarette smokers, has been found to be particularly sensitive to chemotherapy, and there are a number of combination therapies currently in clinical use. Chemotherapeutic agents currently under investigation include camptothecan derivatives, which have been found to inhibit DNA topoisomerase I, and taxol, which is an antitubular agent. While these therapies generally are thought to hold promise for inhibiting cancer cell growth and proliferation in a particular tissue or organ, they do not speak to the issue of cancer metastasis. Metastasis is the mechanism by which cancer cells travel or spread from one area of the body to other, often unrelated, areas, thereby resulting in the development of malignant tumors throughout the body. Tumor metastasis is believed to be one of the leading causes of cancer-related mortality. Since SCLC is a form of cancer that is characterized by early onset of metastatic spread, making it a very difficult cancer to cure, the development of new chemotherapeutics which target invasion and metastasis of malignant cells is a particularly important strategy for combating this type of cancer.
Generally, cancer develops in four principle stages: (1) initiation; (2) promotion of cell growth; (3) invasion and metastasis; and (4) death of the host. Initiation is characterized by hyperproliferation of cells which then continues during the growth stage. During invasion and metastasis, tumors form and the cancer spreads to other tissues. Different forms of cancer respond differently to the variety of treatment protocols, depending upon the type and developmental stage of the cancer. In the case of SCLC, for example, the three primary methods of treatment include surgical removal of cancerous tissue, radiation therapy, and chemotherapy.
Sialyl Lewis X is a cell surface glycoconjugate that serves as a recognition element in cancer metastasis. Structurally, sialyl Lewis X is a tetrasaccharide consisting of N-acetyl neuraminic acid (NeuAc) xcex1-(2xe2x86x923) linked to galactose, which in turn is xcex2-(1xe2x86x924) linked to a glucosamine bearing an xcex1-(1xe2x86x923) linked fucose residue. Sialyl Lewis X is synthesized in the Golgi apparatus, which is located in the cytoplasm of the cell. As illustrated by the following Reaction Equation, 
preparation of sialyl Lewis X begins when an activated sialyl residue (CMP-NeuAc) is transferred to lactosamine using a 2xe2x86x923-sialyl transferase enzyme. Subsequently, activated fucose (GDP-fucose) is transferred to the molecule by a fucosyl transferase enzyme. The resultant tetrasaccharide is then transported out of the Golgi and to the cell surface. Once at the cell surface, sialyl Lewis X serves as a cell recognition element for other molecules, such as selectin proteins (P-selectins), which are on the surface of other cells. Thus, cells having P-selectins at their surface, for example, are able to recognize and adhere to cells which have sialyl Lewis X on their surface. Studies have shown that selectin recognition of sialyl Lewis X at the cell surface is most efficient when both the fucosyl and sialyl residues are present on the sialyl Lewis X molecule. (See e.g., Einhorn L. H., Bond W. H., Hornback N., and Joe B. T. xe2x80x9cLong-Term Results in Combined-Modality Treatment of Small Cell Carcinoma of the Lung.xe2x80x9d Sem. Oncol. 1978, 5, 309-313; Bolscher J., Bruyneel E., Rooy H., Schallier D., Marcel M., and Smets L. xe2x80x9cDecreased Fucose Incorporation in Cell Surface Carbohydrates is Associated with Inhibition of Invasion.xe2x80x9d Clinical and Experimental Metastasis. 1989, 7(5), 557-569; Yamada N., Chung Y. S., Takatsuka S., Arimoto Y., Sawada T., Dohi T., and Sowa M. xe2x80x9cIncreased Sialyl Lewis A Expression and Fucosyl Transferase Activity with Acquisition of a High Metastatic Capacity in a Colon Cancer Cell Line.xe2x80x9d British J. of Cancer 1997, 76(5), 582-587).
An abundance of evidence links the presence of fucosyl and sialyl residues at the surface of cancer cells to both metastatic potential and patient survival. (See e.g., Izumi, Y., Nemoto Y.; Kawamura Y., Nakatsugawa S., Dohi T., Oshima M., Irimura T. xe2x80x9cCorrelation of Cell Surface Sialyl-LeX, Fucosyltransferase Activity and Experimental Liver Metastasis of Human Colon Carcinoma Cellsxe2x80x9d Sixth International Congress of the Metastasis Research Society, Gent, Belgium, Sep. 8-11, 1996. Clinical and Experimental Metastasis 1996, 14(supplement I), 94-95.) This evidence serves to highlight the significance of sialyl- and fucosyl-transferases, the enzymes which mediate the transfer of these residues to cell surface glycoconjugates, as key targets for chemotherapeutic intervention.
Generally, tumor metastasis is facilitated by cell-cell adhesion and communication, which is accomplished through interactions between molecules expressed on the cells"" surface. For example, studies have shown that, during metastasis, many carcinoma cells interact with certain proteins, such as the P-selectins, found on the surface of blood platelets (thrombocytes). (See e.g., Handa K., White T., Ito K., Fang H., Wang S-S., Hakomori S-I. xe2x80x9cP-Selectin-Dependent Adhesion of Human Cancer Cells. Requirement For Co-Expression of a xe2x80x98PSGL-1-Likexe2x80x99 Core Protein and the Glycosylation Process for Sialosyl-Lex or Sialosyl-Leaxe2x80x9d Int. J. Oncol. 1995, 6, 773-81 and references cited therein; Stone J. P., and Wagner D. D. xe2x80x9cP-Selectin Mediates Adhesion of Platelets to Neuroblastoma and Small Cell Lung Cancerxe2x80x9d J. Clin. Invest. 1993, 92, 804-13). This evidence suggests that the ability of malignant cells to bind particular proteins, such as the selecting, mediates the transport of those cells through the body. Thus, if selectin binding by cancer cells can be inhibited or prevented, it is likely that metastasis of malignant cells can be slowed or eliminated. Since the presence of flicose and/or sialic acid on sialyl Lewis X has been shown to facilitate selectin recognition at the cell surface, and since selectin binding has been demonstrated to mediate metastasis, it is highly likely that fucose and/or sialic acid are important recognition elements in tumor metastasis. Thus, if either fucose or sialic acid is prevented from being incorporated into cancer cell surface glycoconjugates, P-selectin binding can be inhibited, and tumor metastasis can be slowed or prevented entirely. Cumulatively, this research indicates that the development of enzyme substrate analogs designed to target and effectively inhibit the transferase enzymes that facilitate the incorporation of either fucose or sialic acid into cancer cell-surface glycoconjugates is a viable strategy for impeding or eliminating these important metastatic pathways.
Enzyme inhibitors also can be used to affect undesirable biochemical processes in the context of human immunodeficiency virus (HIV) infection. The lifecycle of HIV has seven distinct phases: (1) viral binding to host cell receptors; (2) entry of the viral core into host cells; (3) shedding of the viral core; (4) reverse transcription of viral RNA into viral DNA; (5) integration of viral DNA into host DNA; (6) viral DNA replication and protein synthesis; and (7) budding of new viral cores. Current drug therapies for the treatment of HIV focus on two of these phases by inhibiting two key enzymes, HIV-reverse transcriptase (HIV-RT) and HIV-protease.
HIV-RT is an enzyme endogenous to retroviruses that has two known catalytic duties. It functions as an RNA or DNA dependent DNA polymerase and as a ribonuclease H. Currently, only the polymerase activity has been targeted for enzyme inhibition. HIV-RT inhibitors block the transcription of viral RNA into viral DNA. These inhibitors were the first HIV inhibitors approved by the FDA, but viral resistance to their activity tends to develop over time, thereby limiting their use as a sole therapy. The first drug approved for treatment of HIV infection was AZT (3xe2x80x2-azido-3xe2x80x2deoxythymidine), a compound in which the natural 3xe2x80x2-OH of thymidine is substituted with an azide. This azide functionality eliminates the possibility of covalent elongation of a growing DNA strand by functioning as a chain terminating inhibitor. Since the active form of AZT includes a 5xe2x80x2-triphosphate, which is synthesized and added in vivo by endogenous host enzymes after the uncharged form of the compound has been transported across the cell membrane, AZT is properly characterized as a prodrug.
One of the major drawbacks of currently available chain terminating nucleoside inhibitors is the high mutation rate of HIV-RT, which promotes viral resistance to the available inhibitor therapies. A recent study by Harrison and Verdine (Huang H., Chopra R., Verdine G., Harrison S. C. xe2x80x9cStructure of a Covalently Trapped Catalytic complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistancexe2x80x9d Science 1998, 282, 1669-1674) demonstrated that the binding pocket of the catalytic complex of HIV-RT is a 3xe2x80x2-binding pocket and that this pocket incorporates point mutations in those enzymes that are resistant to AZT as well as other nucleoside analog inhibitors. Although extensive research has focused on the use of various 3xe2x80x2-analogs of nucleosides, much less attention has focused on altering the 5xe2x80x2-triphosphate moiety of the natural nucleoside triphosphate substrates.
Another class of approved compounds is HIV-protease inhibitors, which interfere with the processing of viral proteins. Combination therapy of protease inhibitors and RT-inhibitors has proven promising in some cases; however, the search for less toxic and more potent candidates in both of these classes continues. As combination therapy is the most promising approach to HIV treatment to date, identification of other key enzymes that can be targeted for chemotherapeutic intervention is needed.
One of the most recently targeted enzymes for anti-HIV therapy has been the HIV integrase (HIV-IN) enzyme. This enzyme integrates newly formed viral DNA into host DNA by inserting the viral DNA product of the reverse transciptase and viral RNA reaction into the host""s chromosomes. As no cellular counterpart to this enzyme has been identified, it is an attractive target for chemotherapeutic intervention because compounds directed at its inhibition are unlikely to interfere with or affect the functioning of the host system. Catechol-based analogs, which may be exemplified by the following Formula, 
comprise one class of compounds that have been identified as having potent anti-HIV-IN activity. Recent studies based on a large number of these HIV-IN inhibitors have shown that catechol-based inhibitors have several general structural features in common: (1) the presence of two aryl units, (2) separated by a central linker, (3) with at least one aryl ring having an ortho bis-hydroxylation pattern (Zhao H., Neamati N., Mazumder A., Sunder S., Pommier Y., ad Burke T. xe2x80x9cArylamide Inhibitors of HIV-1 Integrasexe2x80x9d J. Med. Chem. 1997, 40, 1186-1194). The inhibitory potency of compounds in this general class has been demonstrated repeatedly, and development of improved catechol-based HIV-IN inhibitors holds promise for chemotherapeutic intervention in the progression of HIV infection (Zhao H., Neamati N., Mazumder A., Sunder S., Pommier Y., ad Burke T. xe2x80x9cArylamide Inhibitors of HIV-1 Integrasexe2x80x9d J. Med. Chem. 1997, 40, 1186-1194; Lin Z., Neamati N., Zhao H., et al. xe2x80x9cChicoric Acid Analogues as HIV-1 Integrase Inhibitorsxe2x80x9d J. Med. Chem. 1999, 42, 1401-1414). Thus, in the field of HIV research as well as cancer research, the successful development of enzyme substrate analogs to inhibit particular target enzymes is a viable strategy for either eliminating or slowing the progression of the disease.
A well-designed substrate analog or competitive inhibitor should be capable of passing through the cell membrane, being recognized by the target enzyme, binding the enzyme successfully, not reacting in a manner that will yield the undesirable biochemical product, and not degrading or hydrolyzing within the environment of the cell. For example, in the case of targeting SCLC tumor metastasis when developing successful competitive inhibitors of sialyl transferase or fucosyl transferase, the inhibitor or analog should structurally resemble the relevant nucleotide sugar substrate in many respects. A nucleotide sugar is the glycosyl or sugar donor in biosynthetic reactions catalyzed by glycosyl transferases. Nucleotide sugar substrates consist of the relevant carbohydrate residue (sialic acid for sialyl transferase and fucose for fucosyl transferase) linked to the relevant activating nucleoside mono- or diphosphate (cytidine and guanosine, respectively). In most cases, the link between the carbohydrate residue and the nucleoside phosphate is either a mono- or a diphosphate linkage, which, in either case, results in the sugar being linked to the other components via at least one phosphodiester bond. Additionally, the 5-bond distance between the sugar and the 5xe2x80x2-nucleoside residue must be maintained, since this distance has been shown to be critical for achieving enzyme-substrate binding. These basic components, that is, the carbohydrate residue, the nucleoside phosphate, and the linking group need to be present in the inhibitor, otherwise the inhibitor cannot be recognized and bound by the target enzyme. However, because the phosphodiester bond connecting the sugar to the nucleoside is a highly labile bond, replacing the oxygen atom linkage in that bond with a carbon atom linkage, creating what is known as a C-glycoside, rather than the natural O-glycoside, often tends to create a more robust analog under physiological conditions. However, incorporation of the carbon atom presents its own set of constraints. In order for a C-glycoside to fit into the enzyme active site, the carbon atom must be incorporated in an equatorial rather than an axial position so that the molecule maintains the appropriate stereochemistry needed for complexation with the enzyme. The challenge, therefore, lies in designing and implementing chemical substitutions within the natural substrate molecule to impart all the above-noted characteristics to an inhibitor.
There are several prior art examples of glycosyl transferase inhibitors, including tunicamycin, a naturally occurring antibiotic which is an isosteric analog of nucleoside diphosphates. However, because tunicamycin is highly toxic, researchers have sought alternative isosteres of this compound. One design strategy has been to produce analogs with a more stable functionality than the extremely labile diphosphate linkage, while still retaining the critical 5-bond distance between the sugar and the 5xe2x80x2-nucleoside residue. To date, both O-linked and C-linked glycosidic analogs of the nucleoside diphosphates have been developed. However, while the prior art glycosyl transferase analogs have been shown to actively inhibit the target enzyme, they have proved to be unsatisfactory in vivo in several regards. First, as with tunicamycin, toxicity may present a problem. Second, the oxygen atom linkages comprised by the molecules frequently result in enzymatic cleavage of the molecule within the cellular environment. Finally, the presence of a formal charge on the molecule often precludes its successful transport across the cell membrane.
In the light of the known disadvantages of prior art substrate analogs, phosphate linkages present in many enzyme substrates have been recognized as likely sites for altering the chemistry of the natural substrate in a manner which would result in a successful inhibitor compound. Depending on the targeted enzyme substrate, these phosphate linkages may be mono-, di-, or triphosphate linkages. Among the reasons for identifying the phosphate linkage as a site for chemical substitution is that the phosphate moiety itself is unsuitable in a substrate analog inhibitor because it is easily hydrolyzed or cleaved by other enzymes present within the cell and, as an ionic or charged moiety, it generally has difficulty in crossing cell membranes, which are lipophilic.
Sulfones have been recognized in the literature as substitutes for phosphate moieties, such as biological phosphodiesters, in a variety of contexts. (Castro A. and Spencer T. A. xe2x80x9cFormation and Alkylation of Anions of Bis(methylsulfonyl)methane.xe2x80x9d J. Org. Chem. 1992, 57, 3996-3499.) In particular, the disulfone moiety has been recognized as a good potential surrogate for the diphosphate group present in a variety of natural enzyme substrates for several reasons: (1) the disulfone moiety is not readily hydrolyzed; (2) disulfones presumably have less difficulty in crossing the cell membrane due to their electrical neutrality; (3) disulfones are generally similar in size and shape to the corresponding diphosphates; and (4) disulfones likely are sufficiently polar to bind the enzyme active site in the place of an ionic diphosphate moiety. (Id.) Despite recognition of the usefulness of disulfones, prior art disulfone reagents and methods of preparing substrate analogs for certain enzymes have been generally unsatisfactory, as the design and preparation of a suitable disulfone reagent capable of being used to synthesize stereospecific substrate analogs in an acceptable yield has proved elusive. For example, the use of gem-disulfones as pyrophosphate analogs was initially investigated by Spencer in his attempt to produce farnesyl transferase inhibitors (Castro A. and Spencer T. A. xe2x80x9cFormation and Alkylation of Anions of Bis(methylsulfonyl)methane.xe2x80x9d J. Org. Chem. 1992, 57, 3996-3499). However, the highly basic di- and tri-anion chemistry employed in Spencer""s syntheses resulted in low yields and produced compounds with no resultant biological activity. Thus, an important reason for the reported unacceptable yields of disulfone analogs is that known reagents and reaction sequences require strongly basic conditions which ultimately degrade the target molecule over the course of the reaction.
However, the disulfone moiety is extremely versatile, and its application in the synthesis of relevant enzyme inhibitors goes beyond its ability to substitute for the phosphate moiety frequently present in natural substrates. For example, in the case of HIV-1 inhibition, the disulfone moiety can be used to synthesize effective HIV-integrase inhibitors by serving as the central linker between the two aryl units of a catechol derivative. Many catechol-based inhibitors have been shown to inhibit HIV-IN effectively in cell-free assays. However, many of these inhibitors exhibit collateral cellular toxicity, which is thought to be caused by either their oxidation to reactive quinone species or, perhaps, catabolism to cytotoxic metabolites. Other catechol inhibitors may be characterized as prodrugs, since they would rely on host enzymes to convert them to their active inhibitory form after they have been transported across the cell membrane in an electrically neutral, inactive form. Still other catechol derivatives would exhibit a short half-life, metabolizing very quickly and affecting free enzyme concentrations for only limited periods of time.
In view of the foregoing, a need exists for suitable reagents and methods of preparing biologically relevant enzyme inhibitors which overcome the shortcomings of the prior art.
In accordance with the principles of the present invention, a disulfone compound is provided. Methods of preparing various disulfone compounds are also provided. Methods of using a variety of disulfone compounds as reagents capable of forming symmetrical and non-symmetrical xcex1, xcex2 unsaturated gem-disulfones are also provided. There is also provided a disulfone reagent which reacts with both aromatic and aliphatic aldehydes in good to moderate yield to give exclusively the trans isomer.
In accordance with further aspects of the present invention, a methodology for stereospecifically preparing potential gem-disulfone enzyme inhibitors is provided. A synthetic design which allows easy substitution of functional groups so that a number of substrate analogs can be synthesized readily is also provided. In this regard, a reagent which is capable of incorporating a disulfone moiety into a sugar nucleoside so that a suitable substrate analog can be synthesized is also provided. Inhibitors of diphosphate-dependent enzymes, such as enzymes which catalyze the metastatic pathways of certain cancers like small cell lung cancer are also provided. In addition, there is provided a new class of compounds which are potential glycosyl transferase inhibitors. Potential chemotherapeutics which will inhibit the incorporation of sialic acid and/or fucose into glycoconjugates present at the surface of certain cancer cells are also provided. Additionally, a class of potent catechol-based enzyme inhibitors, such as HIV-1 integrase inhibitors, is provided.
Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating exemplary embodiments of the present invention, are given for purposes of illustration only and not of limitation. Many changes and modifications within the scope of the instant invention may be made without departing from the spirit thereof, and the invention includes all such modifications.